KR20130099926A - Compounds for the treatment/prevention of ocular inflammatory diseases - Google Patents

Abstract

The present invention relates to compounds of formula 1 and pharmaceutically acceptable salts thereof for the treatment and / or prevention of inflammatory eye diseases, in particular uveitis, severe conjunctivitis, dry eye and diabetic retinopathy.

Description

Compound for the treatment / prevention of inflammatory eye disease {COMPOUNDS FOR THE TREATMENT / PREVENTION OF OCULAR INFLAMMATORY DISEASES}

The present invention relates to a new therapeutic use of the compound represented by the following formula (1).

More specifically, the present invention relates to ocular inflammatory diseases, more specifically uveitis, severe conjunctivitis (vernal keratoconjunctivitis), and dry eye syndrome. ) (Keratoconjunctivitis sicca) or the use of derivatives of these compounds and their pharmaceutically acceptable salts for the treatment and / or prevention of diabetic retinopathy.

Inflammatory eye disease is the leading cause of vision changes in the world.

More specifically, uveitis is associated with inflammation of the uvea, the vascular middle coat of the eye consisting of the iris, ciliary body and choroid. In uveitis, infection is the result of a wide variety of traumatic and immune-mediated insults.

Conjunctivitis includes diseases characterized by swelling, itching or burning feeling, or redness of the conjunctiva, a membrane that covers the white of the eye. Aetiology of conjunctivitis includes infectious and non-infectious conjunctivitis. Conjunctivitis is usually acute in the case of bacterial or viral infections and chronic in allergic cases.

Dry eye is one of the most common eye diseases. It is also called keratoconjunctivitis sicca (KCS). It is characterized by symptoms of eye irritation and can lead to blurred vision, which increases the risk of corneal infection and ulceration. The pathogenesis of eye dryness is not fully understood, but it is widely recognized that it is associated with ocular surface inflammation.

Diabetic retinopathy is the result of chronic hyperglycaemia, leading to capillary lesions with functional alterations such as edema and ischemia. Laser photocoagulation is still the standard of care, and vitrectomy is used in the case of retinal detachment. Lucentis ^® (ranibizumab) is used for the treatment of macular edema.

The main therapeutic choices for subjects with uveitis, severe conjunctivitis and dry eye are made locally or systemically administered corticosteroids. Nevertheless, corticosteroids can cause serious side effects such as cortisone-induced cataract or glaucoma, secondary infections, and delayed wound healing, both through the systemic route as well as through the local route. (side effects)

There are also non-steroidal anti-inflammatory agents such as diclofenac or flurbiprofen. However, many of the subjects are either unresponsive or poorly treated to sterile or non-sterile treatment.

There are also antimetabolite drugs such as methotrexate and azathioprine with hemato- and hepatotoxicity, which are recalcitrant administered by the oral route and It is essential to use in the treatment of very serious uveitis, immunosuppressants such as cyclosporine A and tacrolimus, and also the risk of kidney impairment, lymphocyte proliferative syndrome (lymphoproliferative) many side effects, such as increased risk of syndromes and malign skin diseases. To limit these side effects, these immunosuppressive agents can be used by topical routes, but these compounds do not dissolve in the water medium because of their macrocyclic structures. They are mainly formulated in oil vehicles that have the disadvantage of causing irritant, painful and blurred vision. These compounds are not generally resistant to subjects.

The present invention provides new uses of one or more compounds of formula (1) and their pharmaceutically acceptable salts, in particular in the prevention and / or treatment of inflammatory eye diseases such as uveitis, severe conjunctivitis, dry eye or diabetic retinopathy To overcome the shortcomings from the prior art.

Additionally, the present invention provides an aqueous pharmaceutical composition containing a compound of formula 1, which can reach the posterior chamber of the eye. This means many advances for the treatment of inflammatory eye diseases.

Moreover, the compounds of the present invention have little or no effect on the systemic immune response, thus significantly limiting the potential side effects associated with the administration of such compounds.

The present invention improves clinical signs in uveitis models, especially when the compounds represented by the following formula (1) (hereinafter referred to as "compounds of the present invention") and their pharmaceutically acceptable salts, are administered locally: Based on unexpected results capable of protecting the ocular tissue of the blood ocular barrier and the anterior and posterior chambers without altering the systemic immune response. Likewise, the compounds of the present invention are also useful in severe conjunctivitis, dry eye and diabetic retinopathy.

The preferred effects of tresperimus and ansperimus, obtained in the compounds of the invention and their pharmaceutically acceptable salts, in particular in other pharmaceutical models, are that these compounds may affect T lymphocytes in the eye. It can induce the regulation of the mediated response and macrophage activity.

The compounds of the present invention and their pharmaceutically acceptable salts, which have a linear structure, are soluble and stable in aqueous media, so they can be administered locally in aqueous formulations which are completely biocompatible and do not cause irritation or blurred vision. .

According to a first aspect, the present invention therefore provides the preparation of a medicament useful for the treatment and / or prophylaxis of inflammatory eye diseases, in particular uveitis, severe conjunctivitis, dry eye and diabetic retinopathy, the compounds of the present invention and pharmaceuticals thereof Pertaining to the use of acceptable salts, in particular tresperimus and annisperimus.

According to a second aspect, the present invention provides a method for preventing and / or treating inflammatory eye disease, mainly uveitis, severe conjunctivitis, dry eye syndrome or diabetic retinopathy, wherein the method comprises one or more compounds of the present invention or a pharmaceutical thereof To the subjects in need thereof. In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof is tresperimus or annisperimus. The compound (s) of the invention are administered in eye drops, solutions that can be injected by intraocular or periocular route, or in implantable systems.

The compounds of the present invention and their pharmaceutically acceptable salts, in particular tresperimus and annisperum, are particularly useful for the treatment and / or prophylaxis of uveitis, dry eye or diabetic retinopathy.

According to a third aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof as the only active substance for providing local administration to a subject having ocular inflammation diseases ( to a suitable formulation of a pharmaceutical composition; The present invention more specifically relates to aqueous pharmaceutically acceptable formulations suitable for topical administration.

According to a fourth aspect, the compounds of the present invention, in particular tresperimus and annisperimus, and pharmaceutically acceptable salts thereof, are anti-VEGF agents, anti-TNF agents, corticosteroids, non-steroidal anti It may be administered in combination with an infectious agent, an antibiotic or an immunosuppressant.

Further understanding of the features and advantages of the present invention may be made with reference to the following detailed description and claims.

The compounds of formula 1 according to the invention have little or no effect on the immune system response in the prophylaxis and / or treatment of inflammatory eye diseases such as uveitis, severe conjunctivitis, dry eye or diabetic retinopathy, and thus are potentially associated with the administration of such compounds. It has the effect of significantly limiting side effects.

FIG. 1 is a diagram showing the effect of tresperimus injection on clinical experimental auto-immune Uveoretinis (EAU) in rats.
FIG. 2 shows intravitreal for EAU histopathological score (A) and EAU histopathological changes (C) in rats treated with tresperimus compared to rats injected with saline (B). IVT) A diagram showing the effect of tresperimus injection, where a, b, d, e = photoreceptor layers; c, f = optic nerve heads.
3 shows the effect of tresperimus on Delayed Type Hypersensitivity (DTH) specific to S-antigen in rats treated by intravitreal injection.
4 shows the ocular distribution of tresperimus after dropping 1% solution of eye drops twice daily for 4 days to male New Zealand rabbits.
5 shows the effect of tresperimus after treatment with instillation on clinical signs of uveitis induced by LPS (lipopolysaccharide).
FIG. 6 shows the effect of tresperimus after treatment with a drop on the number of inflammatory cells infiltrated in uveitis induced by LPS.
FIG. 7 shows the effect of tresperimus on the volume of tears measured by the Phenol Red test.
FIG. 8 shows the effect of tresperimus on the stability of the tear film measured by the time of rupture of the tear film.
9 shows the effect of tresperimus on the production of MCP-1 and IL-6 in a vitreous body.
FIG. 10 shows the effect of tresperimus on the magnitude of pseudo-oscillations at different frequencies.

Unless specifically stated, all technical and specific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, the following definitions are provided to assist in understanding the practice of the present invention.

The "subject" is preferably a mammal, more preferably a human.

As used herein, the term “intention used for treatment and / or prevention” can be understood to protect the direct use of the compound or salt thereof for the treatment and / or prevention of the above specified diseases.

"Methods of prevention and / or treatment" may be understood to protect such methods in which a compound or derivative or salt thereof is administered for the treatment and / or prevention of a specified disease.

"Ocular inflammatory diseases" is a generic term for an infection that affects any part of the eye or surrounding tissue. Infections involving the eye include: from allergic hay fever conjunctivitis, from severe allergic hay fever conjunctivitis, to severe conjunctivitis (spring cornea conjunctivitis), uveitis, scleritis, episcleritis, optic neuritis, Keratitis, orbital pseudotumor, retinal vasculitis, dry eye, diabetic retinopathy, and age-related macular degeneration (AMD), eye tissue, ie blindness It can range from potentially rare conditions that can lead to blindness, such as ocular manifestations of systemic disease damage to progressively inducible retinas. The location of the inflammation is managed by a diagnosis for ocular inflammatory disease. Inflammatory eye disease can result from several causes.

According to the present invention, uveitis is non-infectious, from drug-induced trauma, cause of immune mediation, malignant cause, or post-ophthalmic surgery causes. Triggered.

The pharmaceutical compositions of the present invention are also used after ophthalmic surgery, such as cornea transplantation, causing eye inflammation.

"Uveitis" refers to inflammation of the uvea, the middle coat of the eye, including the iris, the ciliary body and the choroid. It is classified by its location, clinical process and its laterality.

"Anterior" refers to the inclusion of the iris, cornea, pupil, aqueous humor or ciliary body. For example, Kawasaki disease may be referred to as anterior uveitis.

"Intermediate" refers to the vitreous, pars plana, peripheral retina and sclera.

"Posterior" is associated with the choroid or retina by dilation of the fovea and optic nerve. Among non-infective posterior uveitis, Behcet's disease, Bogt-Koyanagi-Harada disease, pars planitis, sarcoidosis, idiopathic retinal vasculitis vasculitis and multifocal retinochorioditis may be mentioned.

"Panuveitis" is used when more than one segment is affected.

According to the present invention, conjunctivitis is non-infectious and is often derived from severe ocular allergic reactions that lead to ulcers, which include the risk of significant and crucial blindness.

Allergic conjunctivitis is an inflammatory response of the conjunctiva (the covering of the eye and the inner part of the eyelid). The eye may then be red, sting, burn, itch, scratch and weep. Light is difficult to withstand (photophobia). The eyelids are often red and swollen, with conjunctival swelling (chemosis), or even deep markings of the eye contours or significant mucus secretions. Conjunctivitis rarely affects the cornea. This is more frequent and probably less serious than eye allergies. This type I response is often the result of abundant pollen during the spring and summer periods (tree and grass pollen). The term "allergic keratoconjunctivia" is used when damage is also involved in the conjunctiva as well as the cornea.

Sometimes there are other types that are rarer and more specific, as well as more severe allergic combinations of type IV sensitivity and type I sensitivity. For example, vernal conjunctivitis is a serious form of ocular allergy that sometimes causes ulcers that always include a significant and critical risk of vision loss. These ulcers are often located on top of the cornea, and papillae form on the conjunctiva on the eyelids.

Severe conjunctivitis, such as uveitis, is treated with corticosteroids, non-steroidal anti-inflammatory agents or immunosuppressants.

"Dry eye or dry keratoconjunctivitis or ocular dryness" is associated with all pathologies of the eye resulting from secretion and poor quality of tears by insufficient amounts of lacrimal glands. In the application of the present invention, dry eye also relates to all forms of tear deficiency (including autoimmune Sjogren's syndrome and non-Sjogren tear deficiency) and forms of evaporation. Dry eye is also known as tearing of the tear function unit, which is an integrated system that includes tear glands, ocular surfaces (corneas, conjunctiva and meibomian glands) and eyelids, as well as sensory nerves in contact with them. .

"Diabetic retinopathy" is associated with damage to the microcirculation of the retina and choroid (the damaged organs are the retina, choroid, papilla and iris) because of chronic hyperglycemia. Two forms exist: simple (or non-proliferative) and proliferative.

In some cases, the retina and generally macular edema appear. In other cases, occlusions of the retinal capillaries occur by causing retinal ischemia. Moreover, these two main characteristics can be combined with others leading to periretinal ischemia and macular exudates.

The compounds of the present invention are also useful for the treatment of age-related macular degeneration (AMD).

Compounds of the invention have Formula 1:

here,

n is 6 or 8,

A is a bond, group CH ₂ , group CH (OH), group CHF, group CH (OCH ₃ ), group CH ₂ NH or group CH ₂ O,

R is H or CH ₃ .

A "salt" of a compound of the present invention may be obtained by chemical reaction between the compound of formula 1 and the inorganic or organic acid mentioned below.

Preferred inorganic acids for salt formation are: hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

Preferred organic acids for salt formation are: fumaric acid, maleic acid, oxalic acid, citric acid, trifluoroacetic acid, tartaric acid and sulfur Sulfonic acids (from methanesulfonic acid to dodecanesulfonic acid).

Compound of Formula 1 is:

N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester;

N- [4-[(3-aminopropyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -propanediamide;

N- [4-[(3-aminopropyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -2-hydroxy-propanediamide;

N- [4-[(3-aminopropyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -2-fluoro-propanediamide;

N- [6-[(aminoiminomethyl) amino] hexyl] -N '-[4-[(3-aminopropyl) amino] butyl] -2-methoxy-propanediamide;

N- [6-[(aminoiminomethyl) amino] hexyl] -2-[[[[4-[(3-aminopropyl) amino] -butyl] amino] carbonyl] amino] -acetamide;

N- [6-[(aminoiminomethyl) amino] hexyl] -N '-[4-[(3-aminopropyl) amino] butyl] -ethanediamide;

N- [8-[(aminoiminomethyl) amino] octyl] -N '-[4-[(3-aminopropyl) amino] butyl] -ethanediamide;

N- [8-[(aminoiminomethyl) amino] octyl] -N '-[4-[(3-aminopropyl) amino] butyl] -propanediamide;

N- [8-[(aminoiminomethyl) amino] octyl] -N '-[4-[(3-aminopropyl) amino] butyl] -2-hydroxy-propanediamide;

N- [8-[(aminoiminomethyl) amino] octyl] -N '-[4-[(3-aminopropyl) amino] butyl] -2-fluoro-propanediamide;

N- [4-[(3-aminopropyl) amino] butyl] -2-methoxy-N '-[8-[(aminoiminomethyl) amino] -octyl] -propanediamide;

N- [8-[(aminoiminomethyl) amino] octyl] -2-[[[[4-[(3-aminopropyl) amino] butyl] -amino] carbonyl] amino] -acetamide;

N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[8-[(aminoiminomethyl) -amino] octyl] amino] -2-oxoethyl ester;

N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -ethanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -propanediamide;

2-[[[[4-[(3-aminobutyl) amino] butyl] amino] carbonyl] amino] -N- [6-[(aminoiminomethyl) amino] hexyl] -acetamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -2-hydroxy-propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -2-fluoro-propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[6-[(aminoiminomethyl) amino] hexyl] -2-methoxy-propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[8-[(aminoiminomethyl) amino] octyl] -ethanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[8-[(aminoiminomethyl) amino] octyl] -propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[8-[(aminoiminomethyl) -amino] octyl] amino] -2-oxoethyl ester;

2-[[[[4-[(3-aminobutyl) amino] butyl] amino] carbonyl] amino] -N- [8-[(aminoiminomethyl) amino] octyl] -acetamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[8-[(aminoiminomethyl) amino] octyl] -2-hydroxy-propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[8-[(aminoiminomethyl) amino] octyl] -2-fluoro-propanediamide;

N- [4-[(3-aminobutyl) amino] butyl] -N '-[8-[(aminoiminomethyl) amino] octyl] -2-methoxy-propanediamide;

And pharmaceutically acceptable salts thereof.

More preferred compound of Formula 1 is:

N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester (trespermus) And

N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester, and pharmaceuticals thereof Is selected from acceptable salts.

Particularly preferred compounds of formula 1 are N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2- Oxoethyl ester, tri-hydrochloride and N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl ] Amino] -2-oxoethyl ester, tetra-hydrochloride.

Pharmaceutical compositions of the present invention, usually in combination with one or more pharmaceutically acceptable carriers or excipients, may be used as a sole active substance to form a compound of the present invention or a pharmaceutically acceptable salt thereof. Include.

"Pharmaceutical carrier" refers to an administrable pharmaceutically acceptable excipient of the active substance or a mixture of various pharmaceutically acceptable excipients. They can promote or enhance the preparation of the composition and can stabilize the composition. Moreover, pharmaceutically acceptable carriers can enhance the effectiveness of the composition, enhance the ocular resistance of the active substance, and modify its release profile.

They must also be compatible with other components of the composition, both pharmaceutically and physiologically acceptable in the biocompatible, non-toxic sense. Such carriers may be taken in a variety of forms depending on the form of the preparation for the desired administration, for example, topical administration.

"Local administration" can be understood as all ocular routes, ie topical and injectable administration, and by means of implantable systems.

“Topical administration” can be in the form of, for example, eye drops, eye drops or ocular instillation, sprays, creams, ointments, gels ( non-limiting forms of gels, hydrogels, oleogels, hydrophilic lenses, inserts, and implants. Dosage forms include, for example, solutions, suspensions, colloidal systems (eg, liposomes, emulsions, microemulsions, nanoemulsions, microparticles, nanoparticles, microspheres, niosomes). ), Dendrimers), micelles, mixed micelles, complex systems such as cyclodextrin solutions, as well as non-transplantable insertions in the form of But not limited to, and may be a non-limiting form of discs, films or strips.

"Injectable administration" includes intraocular (intravitreal, IVT), periocular, including subconjunctival, sub tenon's, retrobulbar, and intrascleral administration. May be a non-limiting method.

"Intrascleral administration" can be performed in an injectable or implantable system. Dosage forms include, for example, biodegradable or non-biodegradable implants, as well as solutions, suspensions, colloidal systems (eg liposomes, emulsions, microemulsions, nanoemulsions, microparticles, nano Particles, microspheres, niosomes, dendrimers, micelles, mixed micelles, but are not limited to, rods, nails ( nails, may be a non-limiting method of pellets.

In the application of the invention, when the concentration is expressed in m / V, the density of the solution is considered to be one.

Depending on the compound to be delivered, for both routes of administration (local and injectable), most of the above described dosage forms can potentially increase the residence time of the active principle on the surface or vitreous of the eye and It can provide slow and sustained release of the encapsulated compound, avoid toxicity and increase eye tolerance.

According to the present invention, for the treatment and / or prophylaxis of inflammatory eye diseases, in particular uveitis, severe conjunctivitis, dry eye or diabetic retinopathy, the compounds of the present invention or their pharmaceutically acceptable salts are based on aqueous pharmaceutically It may be administered via an acceptable composition or formulation suitable for topical administration, preferably via drops, or by injectable administration, preferably intravitreal administration.

For topical or injectable administration, the excipient must be pharmaceutically acceptable and appropriate for this type of ocular administration.

The aqueous medium used in the present invention consists of water which does not contain inhibitors, physiologically and ophthalmologically. The pharmaceutical composition of the present invention is in the form of an aqueous formulation having a pH that is biophysically compatible for the ocular pathway. "Physiologically compatible pH for said ocular pathway" means a pH in the range of about 5.5 to about 8, preferably in the range of about 6.0 to about 7.5. The pH of the formulation may be, for example, acids such as acetic acid, boric acid, lactic acid, hydrochloric acid; Salts such as, for example, sodium hydroxide, sodium borate, sodium citrate, sodium acetate; Or a pharmaceutically acceptable buffer solution such as, for example, sodium phosphate buffer, potassium phosphate buffer, sodium citrate buffer. Aqueous formulations of the present invention are isotonic and physiologically applied for ocular, topical and intraocular administration. The osmotic pressure of the formulation is close to the physiological pressure and generally comprises between about 200 mOsm and about 400 mOsm, preferably between about 260 and about 340 mOsm. If necessary, the osmotic pressure can be adjusted using appropriate amounts of physiologically and ophthalmically acceptable excipients. Sodium chloride is generally used as tonicity agent at concentrations (in m / V) not exceeding 0.9%. One or more salts of equivalents composed of anions and cations may also be used. Depending on the therapeutic indication of the present invention, the osmotic pressure can be selectively corrected by optionally adding sugar or polyol alone or in combination. The formulations of the present invention have a variety of viscosities from about 0 to about 2000 Centipoises, preferably up to 100 Centipoises, and more preferably 30 Centipoises.

The composition of the present invention may comprise a reagent that increases the viscosity, thereby extending the precorneal dwelling time of the active source after the drop. These viscosifying agents also have mucoadhesive properties. Mucoadhesive polymers capable of non-covalent bonds with glycoproteins predominantly in the conjunctiva may be used to locally and potentially increase the bioavailability of the ocular of the active source to locally limit formulation to the eye. It can be used in the present invention to optimize the parasitic time of the formulation and thereby reduce the frequency of administration which improves the therapeutic complaints. These polymers are generally macromolecular hydrocolloids. They may also be used alone or in combination in the present invention, for example, methylcelluloses, sodium carboxymethylcelluloses, hydroxyethylcelluloses, hydroxypropylcelluloses, hydroxy Cellulose derivatives such as hydroxypropylmethylcelluloses; Acrylic derivatives such as, for example, polyacrylic acid salts and functional derivatives thereof (or polycarbophils); Carbomers; Natural products such as, for example, alginates, chitosans, pectins, hyaluronic acid and derivatives thereof; Polysaccharide derivatives such as, for example, gellan gum and derivatives thereof, xanthan gum, carrageenans; Co-polymers such as poloxamers. Polymers that themselves have a gelling capacity can be included in the preparation of the pharmaceutical compositions of the present invention. So-called phase transition systems are liquid and lead to the formation of gels compatible with ocular function by ionic activity depending on the pH and temperature. For example, polymers such as polyacrylic acid derivatives, cellulose derivatives, methylcellulose, copolymers and poloxamers can be cited.

The compositions of the present invention may also contain excipients well known to those skilled in the art, for example, surfactants, co-surfactants, co-solvents, penetration agents, gelling agents ( gelling agents, emulsifiers, antioxidants, preservatives, and polymers for sustained release.

The pharmaceutical compositions may also be in the form of insertions or solid implants that allow for ocular administration and sustained release of the active source. For example, the formulation of the insert can be performed using a water-soluble polymer. Inert polymers with biocompatibility for the ocular pathway, used for formulation of the appropriate insertion for the ocular pathway, are synthetic, semi-synthetic or natural resources. Compositions of solid grafts may also contain synthetic, semi-synthetic or ternary polymers, preferably polyvinyl alcohols, polylactic-co-glycolic acids, poly-epsilon It may consist of biologically degradable polymers such as caprolactones (poly-epsilon caprolactones), hyaluronic acid esters. These biologically degradable polymers can also be used to encapsulate the active source in microspheres, nanospheres or nanocapsules dispersed in an aqueous solution to provide sustained and desired release of the active source.

Other matrices such as water-soluble lenses that impregnate or contain the active source may increase the parasitic time of the active source on the surface of the eye.

The principles for the preparation and sterilization of these formulations are well known in the art of dosage form technology.

According to a first preferred embodiment, the pharmaceutical composition in the form of eye drops or injectable solutions provides an effective dosage of a compound of the present invention, such as tresperimus, for example, dissolved in a physiological aqueous solution as the main carrier. Include. Such a solution is preferably an aqueous sodium chloride solution, or preferably at a concentration of less than 0.9% (m / V), to achieve the tonicity of the pharmaceutical composition comprised between about 260 and about 340 mOsm. Aqueous glycerol solution at a concentration of less than 2.5% (m / V). This solution is adjusted to a pH close to 6.5, for example with sodium hydroxide. Preferably bioadhesive polymers, such as hyaluronic acid or derivatives thereof, are added. Such dosage forms are preferably sterilized by gamma radiation. Such dosage forms may be in the form of single dose packs.

According to another preferred embodiment, the injectable solution for administration as a biodegradable implant comprises at least an effective dose of a compound of the invention, preferably encapsulated in micro- or nanoparticles, preferably consisting of poly-epsilon caprolactone. do.

The main advantage of the present invention is that all ocular tissues (front or rear) are exposed to the compounds of the present invention, for example upon administration of a pharmaceutically acceptable aqueous composition. In the evaluation of the study of eye distribution of tresperimus in male New Zealand rabbits (FIG. 4) after dropping 1% solution of eye drops twice daily for 4 days, the retina / choroid (posterior chamber) and ciliary / The iris (anterior chamber) is exposed to tresperimus at levels of 0.5-0.7 μM and 0.3-0.7 μM for 24 hours after repeated eye drops twice daily with a simple 1% aqueous solution. Thus, in addition to other topical administrations, such as intraocular injections or transplants, and in addition to better reception, topical administration of the compounds of the present invention allows for much better control of the active substance concentration in ocular tissues. Moreover, plasma levels are observed low (<5 ng to 2 h post-drip) for a short period below the lower limit of quantification (Blq) (after 2 ng / mL 4 h dosing) after the drop of eye drops. This allows to avoid unwanted immunosuppressant system influences.

The concentration of therapeutically active substance in the formulation for the intravitreal route may vary from 0.1 μM to 100 mM, preferably 1 μM to 10 mM, and more preferably 10 μM to 0.1 mM. The concentration of therapeutically active substance in the formulation for ocular topical drops may vary from 0.001% to 5% (expressed in m / V), preferably from 0.001% to 1.5%, more preferably from 0.01% to 1.5%. . Such concentrations may be applied for other ocular topical routes of administration and may vary depending on the therapeutic indication. Route of administration and dosage are at the discretion of the physician depending on the subject, symptoms and severity of the illness.

Such compositions are prepared by any process for preparing dosage forms well known in the pharmaceutical art.

According to another aspect, the compounds of the present invention may be combined with other therapeutic agents or used in combination. For example, a subject may be treated with tresperimus and / or aniserimus in combination with one or more compounds of the present invention or pharmaceutically acceptable salts thereof, particularly other conventional medicines for the treatment of inflammatory eye diseases. Various active agents can be administered simultaneously, sequentially or over a period of time. It may be desirable not to administer the compounds of the present invention or pharmaceutically acceptable salts thereof in combination with Lck enzyme inhibitors.

According to some embodiments, the present invention thus provides, for example, corticoids such as dexamethasone, prednisolone, and triamcinolone; For example, cyclophosphamide, methotrexate, azathioprine, cyclosporine A, tacrolimus, sirolimus, myoliphenolate mofetil immunosuppressants with an active mechanism from compounds of the invention, such as (mycophenolate mofetil); Of uveitis, selected from anti-TNF agents, such as, for example, rituximab, dacilizumab, infliximab, adalimumab, and etanercept A pharmaceutical composition comprising as an active substance a pharmaceutically acceptable salt thereof or at least one compound of the formula (1) in combination with one or more drugs used for treatment.

According to some embodiments, therefore, the present invention is directed to, for example, corticosteroids such as dexamethasone, prednisolone; Non-steroidal anti-inflammatory agents such as nedocromil, liodoxamide, olopatadine; Antibiotics, antifungal and antibacterial agents such as tobramycine, natamycin and moxifloxacine; For example, at least one drug in combination with one or more drugs used in the treatment of severe conjunctivitis, selected from immunosuppressive agents having a mechanism different from the active mechanism of the compounds of the invention, such as cyclosporine A, tacrolimus, sirolimus. The present invention relates to a pharmaceutical composition comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active substance.

According to another embodiment, the present invention provides an immunosuppressive agent having a mechanism different from the active mechanism of the compound of the present invention, such as, for example, cyclosporin A and mycophenolate mofetil; For example, corticosteroids such as loteprednol, rimoxelone and fluorometholone; And at least one compound of the present invention or a pharmaceutically acceptable salt thereof in combination with one or more drugs used in the treatment of dry eye, selected from tetracyclines. They can also be used in combination with artificial tears and secretogogues.

According to another embodiment, the present invention provides anti-VEGF agents such as, for example, ranibizumab, pegatapnib, bevacizumab; Anti-TNF agents such as, for example, Rituximab, Dasilizumab, Infliximab, Adalimumab and Itanercept; Corticosteroids such as, for example, dexamethasone, prednisolone and triamcinolone; And one or more drugs used in the treatment of diabetic retinopathy, for example selected from immunosuppressive agents having an active mechanism different from the compounds of the invention such as, for example, cyclosporin A, tacrolimus, everolimus and sirolimus. A pharmaceutical composition comprising as an active substance at least one compound of the invention or a pharmaceutically acceptable salt thereof in combination with They can also be used in combination with laser therapy (photocoagulation).

Hereinafter, the present invention will be described in more detail with reference to tresperimus, but those skilled in the art will understand that the present invention is not limited to the compound of Formula 1.

The following examples and embodiments are only intended to illustrate the invention, and various modifications or changes made in consideration of the above embodiments and embodiments may be presented to those skilled in the art, and this is not necessarily the spirit and scope of the present application and the appended claims. It is to be understood that it is included in the claims. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

practice  Yes  1: uveitis

The eye is an immunologically isolated site; However, diseases of the eye resulting from imbalances in the immune system are responsible for and contribute to vision disorders that can lead to vision loss. Animal models, primarily experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (EIU), can be considered as relevant clinical models of eye disease and are immunologically capable of controlling disease in humans. It is a valuable way to study the mechanism:

EAUs derived from rats immunized with purified retinal antigens, mainly S-antigens (S-Ag), develop new clinical strategies for uveitis and relevant clinical models for studying the mechanism of posterior uveitis in humans. Is considered;

EIU is a model of spontaneously releasing severe inflammatory uveitis, including components of the natural immune system. This is a useful model for studying the local view of eye inflammation and is considered a relevant model of anterior uveitis in humans.

In the present invention, the inventors first confirmed that the local ocular administration of the compounds of Formula 1 and their pharmacological salts has a great advantage in two experimental models that are considered as relevant clinical models of uveitis in humans.

remind EAU  Model

The EAU model is intended to understand the association of CD4 + (cluster 4 of differentiation) lymphocytes of macrophages and pro-inflammatory cytokines in physiopathological mechanisms, especially in the mechanism of retinal rupture. Help.

EAU has many clinical and histopathological features with human uveitis, such as sympathetic ophthalmia, birdshot retinochoroidopathy, Bognt-koyanagi-Harada disease, Behcet's disease, and sarcoidosis. Is an inflammatory disease model that shares. This is a clinically relevant model for human eye inflammation.

EAU is induced by immunization with S-antigen (S-Ag), a purified retinal autoantigen also recognized in subjects with uveitis. EAU depends on CD4 + Th1 (interferon-gamma producing cells) and CD4 + Th17 (interleukin-17 producing cells) effector cells, each of the effector phenotypes can induce a pathological response.

However, IL-17 (interleukin-17) plays a dominant role in EAU induced by the interphotoreceptor retinoid-binding protein (IRBP). Neutralization of IL-17 prevents or restores its progression. In addition, Th17 effector cells induce EAU in the absence of interferon (IFN) -gamma.

Macrophages and microglial cells then amplify the response locally and induce rupture of photoreceptors and the retinal tissue. Monocytes / macrophages as well as neutrophils are important as effector cells in the EAU, while T-cells act more to initiate and maintain the response. Macrophages cross the blood-retinal barrier and penetrate the retina, while release of mediators such as NO (nitric oxide) and TNF (tumor necrosis factor) can cause severe retinal damage and, consequently, vision in the subject. Can be lost.

We examined the effect of local administration of tresperimus on ocular and immune system responses induced by EAU and S-Ag immunization.

Materials and methods

I. Induction of EAU in Lewis rats

8-week-old female Leuette (R. Janvier, Le Genest Saint Isle, France) was previously described (de Kozak Y, Sainte-Laudy J, Benveniste J and Faure JP.Eur J Immunol. 1981; 11: 612- 617) systematically immunized with purified 40 mg of retinal autoantigen S-antigen (S-Ag).

II. Processing protocol

The administration of Tresperimus is performed by intravitreal (IVT) injection (5 μl) in both eyes 6, 9 and 12 days after S-Ag immunization. At the end of the experiment, ie 19-20 days after immunization, the rats are anesthetized by peritoneal injection of pentobarbital (Sanofi-Aventis, France) before blood collection by heart stab. The let is then euthanized with a pentobarbital lethal dose and both eyes and blood samples are collected for analysis.

In a first experiment, one group of letts was treated with substantially isotonic and sterile saline containing 9 mM tresperimus to achieve 1 mM final solution in the vitreous, and the control group of letts was treated with vehicle (saline) and However, one control group of rats was not treated. Animals are clinically tested with a slit lamp until 9 days after S-Ag immunization until euthanasia. Histopathology of the eye is performed and immunostaining proceeds on sections obtained with cryostats. Inguinal lymph nodes consist of RT-PCR analysis of cytokines.

In the second experiment, one group of letts injected trespermus three times with vitreous and the control letts injected with saline. The rats were subjected to clinically observed and subjected to Delayed Type Hypersensitivity (DTH) analysis. Tresperimus levels in plasma and ocular tissues are measured at 1 h, 3 and 8 days after three injections.

III. Evaluation of EAU Severity

1. Clinical Evaluation

Animals are tested with slit lamps daily until euthanized from 7 to 11 days to assess onset time and severity of disease. The intensity of the clinical ocular inflammation is recorded on a scale from 0 to 7 for each eye as previously described (de Kozak Eur J Imm 2004).

2. Histopathology

At the time of euthanasia (19-20 days after immunization), the eyes of the extracted rats were fixed, processed, and the paraffin sections were cut to haematoxylin-eosin-safran for histological evaluation. It is dyed with. Sections are tested and recorded according to the following scale of semi-quantification of 0-7: (0) no tissue destruction, (1-2) of outer segments of rods and cones Rupture, (3-4) rupture of the outer nuclear layer, (5-6) rupture of the inner nuclear layer, and (7) rupture of the ganglion cell layer.

3. Immunohistochemistry

Eyes (2 eyes / group) are collected, cryostat sections are cut (10 μΜ) and stained for immunochemistry as described above for 10-20 days after immunization. The following antibodies are used: anti-NOS-2 first antibody (Beckton Dickinson Biosciences, Transduction laboratories, San Jose, USA); Wherein -NF-kB / p65 antibody first and then the second antibody Alexa the second antibody coupled with ^{Fluor ® 488 (Molecular Probes, Eugene} , OR); Anti-macrosialin CD68 first antibody (clone ED1) (Serotec, Oxford, GB) followed by a second antibody bound to Alexa 564 (red). Sections are labeled with fluorescence photomicroscopy (FXA; Microphot; Nikon, Melville, Micrographs observed and digitized by NY) are obtained with a digital camera (Spot; BFI Optilas, Evry, France).

IV. Immune response evaluation

1. Delayed type hypersensitivity reaction

DTH is assessed by ear assay by measuring specific anti-S-Ag response 18 days after immunization. Lett measures the sensitivity to 10 mg of S-Ag in the right ear and saline in the left ear. Specific ear swelling is measured 24 and 48 hours after sensitization and yields a difference in thickness (mm) between the two ears.

2. RNA isolation, reverse transcription PCR in lymph nodes and eye cells

Total RNA is isolated from cells collected after centrifugation of aqueous humor / vitre from 19-20 days of immunization, lymph node draining immune site, and each group of eyes.

V. Statistical analysis

Data is expressed as mean ± standard error of the mean (SEM). Clinical and histological evaluations of EAU and DTH are compared using a non-parametric Mann-Whitney U test and are accompanied by a Bonferroni multiple comparison test. The p-value adjusted by the multiple comparison test is calculated for each experiment.

VI. result

1.Pharmacokinetics of Tresperimus in Eye Tissues and Plasma After Intravitreal Injection

Trespermus concentrations in plasma, waterproof / vitreous and retinal / choroidal membranes after intravitreal injection of tresperimus in Lewis let are reported in Table 1 below:

Effect of intravitreal injection of tresmus on EAU histopathology 19-20 days after S-Ag immunization (M ± SEM) Tresperimus concentration
(N = 6) Immunized Lett
(3 IVT injections) Post-injection time 1 hours 3 days 8 days Waterproof / glass
Average 270 2.2 1.8 SEM 61 One One Retina / choroid
(μM) Average 155 36 11 SEM 25 4 4 plasma
(μM) Average 0.11 blq blq SEM 0.03 - -

Blq: lower limit quantification (6 ng / mL) or less

After intravitreal injection of tresperimus, the plasma level of the test sample is quantified only at the first time point 1 hour post-injection, at a low average concentration of about 0.1 μM (about 40 ng / mL). Eye tissue is highly exposed to tresperimus, with a significant content (> 10 μM) in the retina / choroid of 8 days post-injection.

2. Intravitreal injection of tresperimus is an effective treatment of EAU; Clinical observation

Treatment with tresperimus was performed in rats injected with saline (13 days: * p <0.02; 14-19 days: *** p <0.0006), or rats not receiving any intraocular treatment (12 days: * p <0.02). Day 19: *** p <0.0006) (in comparison with FIG. 1) induces a significant reduction in clinical severity of EAU from day 13 post immunization. The severity of the disease is significantly reduced by treatment up to 19 days after immunization, and intraocular treatment appears to be very effective.

3. Intraocular injection of tresperimus protects the retina from rupture and modulates macrocrophage activity.

Rats treated with three injections of tresperimus received lesions (mean EAU severity rating: 3.25 ± 0.5, n = 10) (FIG. 2A) obtained from control rats injected with saline, and any intraocular treatment. Very low grade histological EAU (mean EAU severity rating: 1.45 ± 0.26, n = 10, p = 0.007) compared to the untreated rats (mean EAU severity rating: 3.15 ± 0.6, n = 10, p = 0.08). The mean EAU histopathological record is based on retinal alterations.

Histopathological examination of the retina from saline-injected control rats (FIG. 2B) shows severe anterior uveitis, inflammatory cells (b) with extensive rupture of the photoreceptor cell layer (a, b, white asterisk). Arrows indicate infiltration of the subretinal space and fibrin exudates in the vitreous (arrowhead). Many infected cells are present in the vitreous at the optic nerve head level (arrow) (c). On the other hand, in rats treated with tresperimus (FIG. 2C), the photoreceptor cell layer is largely spared from rupture (e, white asterisk) or the outer segment (d, arrow) due to infiltration of the choroid by inflammatory cells (d, arrowheads). ) Shows partial loss. Inflammation is not visible at the optic nerve head level (f, arrow).

As shown by immunostaining in saline-injected control rats, many ED1-positive giant cells and lymphocytes are mainly cytoplasmic and NF-kappaBp65 in the vitreous, where numerous infiltrations are visible by inflammatory cells. Express nuclear expression. In contrast, in rats treated with tresperimus, slight infiltration by inflammatory cells is visible in eye tissues showing only a reduced number of cells infiltrated in eye tissues and media and cytoplasmic expression of NF-kappaBp65.

4. Intravitreal injection of tresperimus has no effect on the immune response system in vivo.

a) cytokines in inguinal limb fractures (RT-PCR)

The levels of TNF-alpha, IL-2, IFN-gamma and IL-17 are not detected as they are not detected in inguinal lymph nodes from treated and control rats indicating that the treatment has no systematic effect.

b) delayed-type hypersensitivity

DTH is examined by ear analysis to determine specific anti-S-Ag responses. Letts treated with tresperimus demonstrated that T-cell responsiveness to S-Ag in vivo was not reduced by treatment with tresperimus, and that the treatment did not have systemic effects (FIG. 3). There was no significant reduction in ear swelling at 24 and 48 hours compared to control rats treated with IVT injection.

In conclusion, injection of tresperimus in the posterior pole of the eye, the posterior zone of the ciliary body, enables its spread in the anterior and posterior segments of the eye, as has shown its effect in anterior and posterior ocular inflammation in the EAU. do. Moreover, low levels of tresperimus (<90 ng / mL) are found in the plasma without any effect on the immune system response. Indeed, the effects of tresperimus are limited to the eye identified as having no effective diffusion occurring in the general circulation.

We found that three intravitreal injections of tresperimus after immunization with S-Ag during the afferent phase of the disease (6, 9, 12 days) reduce clinical eye inflammation and protect the photoreceptors of the retina. Effective for

In experimenting with the level of Tresperimus action, delayed-type hypersensitivity (DTH) to S-Ag, as assessed by the ear test, does not alter the response of systemic T-cells to S-Ag. No difference from control rats and treated rats (FIG. 3).

Moreover, we show that ocular treatment is ineffective in immune system responses. In fact, at the inguinal lymph node drainage immunization site, the level of inflammatory cytokines such as TNF-alpha, and the levels of cytokines produced by T lymphocytes such as IL-2, IFN-gamma and IL-17, are tresperimus. As long as it is not changed by treatment.

EIU model

The endotoxin-induced uveitis model is a motel of actual ocular inflammation in rats or mice, induced by systematic or local injection of lipopolysaccharide (LPS) of Gram-negative bacteria. This is a model of the actual anterior uveitis in humans, often associated with systemic disorders such as Crohn's disease, ankylosing spondylitis and Blau syndrom.

EIU is characterized by rupture of the ocular brain barrier, intraocular penetration of inflammatory cells into the posterior and anterior segments of the eye, and NO by infiltrated inflammatory cells, mainly macrophages and polymorphonuclear leukocytes (PMNs). And the production of inflammatory cytokines and chemokines, and ocular cells of vascular endothelium, retinal pigment epithelium, microglia and Muller's cells. Although such inflammatory uveitis resolves spontaneously within a few days upon intervention of the natural immune system, it is an important source of damage to eye tissue.

We tested the effect of tresperimus by dropping drops at different concentrations in this EIU model of the let.

Materials and methods

I. Induction of Uveitis by Endotoxin

An 8-week-old female Lewisette (R. Janvier, Le Genest Saint Isle, France) weighing about 250 g is used in this study and 200 μg of Salmonella typhimurium (Sigma) in 0.1 mL of sterile water is applied to these soles. Injected with LPS.

II. Treatment protocol

Tresperimus is applied by drops to each eye twice daily for 4 days with drops of 5% (m / m) and 0.5% (m / m) in a 0.1% (m / m) aqueous solution of sodium hyaluronate. Administered.

On day 3, LPS is administered to the soles of the feet, and after 24 h Tresperimus is administered only one last time. The animals are then tested with slit lamps and their blood is collected and they are sacrificed. The eye is then collected for analysis.

III. Clinical trial

Animals are tested with LPS administration 24 hours after slit lamp, corresponding to the peak of uveitis clinical inflammation. The intensity of inflammation is recorded as follows on a scale of 1 to 6 for each eye, as previously described (De Kozak Y. et al., J. Neuroimmunol. 1998; 86 (2): 171-181). Becomes: 0, no signs of inflammation; 1, isolated inflammation of the iris and cornea; 2, dilation of the vessels of the iris and cornea; 3, hyperemia of the iris associated with the Tindall effect in the rear; 4-6, grade 3, similar signs but with the presence of synechia, fibrinoid exudation or hypopyon. If the grade is above 1, it is considered positive.

IV. Histopathology and counting of inflammatory cells

After euthanizing the animals (ie 24 hours after LPS injection), the let eyes are extracted, then fixed and processed. Paraffin sections are cut for histopathology evaluation. Infiltrated inflammatory cells are stained with haematoxylin-eosin-saffron for histopathology evaluation and then counted sections made on anterior segments of the eye (5 sections per eye). The number of cells is expressed as total number ± SEM mean of cells in each eye and each animal as described above (de Kozak Y. et al, IOVS 1999 Sep; 40 (10): 2275-82).

V. Statistical analysis

Results are shown as mean ± SEM and are compared using the Mann-Whitney U test. P <0.05 is considered statistically significant.

VI. result

The effect of tresperimus is assessed by the EIU model in the let. Acute and chronic ocular inflammation induced by LPS injection is characterized by the infiltration of inflammatory cells 4 hours after injection. It reaches a maximum between 18 and 24 hours and disappears after 4 days.

Tresperimus is injected by infiltration twice daily for 4 days at 5% (m / m) and 0.5% (m / m) in 0.1% aqueous sodium hyaluronate solution. The results (FIG. 5) are represented by clinical score ± SEM for each eye.

In comparison to control animals, treatment with tresperimus shows a significant reduction in eye inflammation obtained (p = 0.001 and p = 0.0001, respectively).

To confirm the clinical effect observed with tresperimus, the total number of cells present in front of the eye is counted, which indicates that the infiltration of inflammatory cells is associated with control animals (average number / section of cells: 12.2 ± 0.8, n = 17 sections). 6, apparently significantly reduced (average number / section of cells: 7.7 ± 0.9, n = 13 sections, p = 0.003 and 7.3 ± 0.7, n = 13 sections, p = 0.0004), as shown in FIG. appear.

These results explain the infiltration of tresperimus in the eyes of Lett by the reduction of ocular inflammation in the model of etotoxin-induced uveitis. The above results indicate that tresperimus treats clinical signs of uveitis by infiltration of eye drops and more generally severe conjunctivitis because these pathologies are mainly treated by corticosteroid and immunosuppressive activity in the pharmacological models of these two animals. It means that it is possible to cure.

Example 2: Dry Eye

In recent treatments, a palliative is essential and the purpose is to replace or maintain the tears of the subject by applying artificial tears frequently. Severe ocular dryness, characterized by severe corneal damage, which increases the risk of secondary infection, can sometimes be treated by anti-inflammatory treatment.

Several animal models have been developed to reflect the different pathophysiological mechanisms involved in KCS. The effect of tresperimus is studied in a mouse model of dry eye using pharmacological inhibition of tear production leading to epithelial changes of the eye surface similar to human KCS, which is exacerbated by dry environmental stress.

Dry eye is induced in mice by a combination of scopolamine, which blocks muscarinic cholinergic receptors in the lacrimal glands, and by placing rats in an extractor hood that reduces humidity and increases air flow. The production and volume of aqueous tears, tear removal, and corneal barrier function are evaluated before treatment and then twice a week after treatment. The results are compared between a group of untreated control rats, and a group of rats placed in the extractor hood, a group treated or not treated with tresperimus, treated with an anticholinergic scopolamine.

An experimental model inducing dry eye induces epithelial changes on the ocular surface, modified corneal epithelial barrier function, reduced density of conjunctival goblet cells, and increased conjunctival epithelial proliferation with fluorescein stained corneas. . This animal model simulates the evaporative components of aqueous-deficient and human dry eye.

Materials and methods

I. Induction of dry eye with cholinergic receptor blockade and desiccation in extractor hood

Male 129SV / CD-1 mice were used in the study and injected subcutaneously with 200 μl of scopolamine three times in 2.5 mg / mL saline for 21 days. The mice are placed in the extractor hood (humidity <50%) during the entire experiment.

II. Aqueous Tear Production

Tear production (PRTT) is measured with cotton yarn infiltrated with phenol red (Zone-quick; Menicon, Japan) applied to the ocular surface of the lateral canthus for 60 seconds. The wetting of the yarn is measured in millimeters, using a scale of cotton yarn.

III. Stability of tear film

The tear film stability test (TBUT) is used to assess the dryness of the eye by measuring the elapsed time between full winks and the development of the first signs of dry spots in the tear film.

One microliter of 0.1% sodium fluorescein is applied to the conjunctival bag and the time (seconds) after the drying point appears is measured after three winks. After 90 seconds, damage to the corneal epithelium is measured and photographed with a slit lamp biomicroscope using cobalt blue light. Clinical scores are recorded using the Draize scoring scale.

result

Tear volume is measured for 3 weeks in C57B16 mice using the Red Phenol test. The results reported in FIG. 7 are expressed as mean tear volume (millimeters) ± standard error of the mean (SEM). They show a dramatically reduced tear volume for 2 days after subcutaneous injection of scopolamine. Dropper of tresperimus twice a day at a dose of 1% in a 0.1% sodium hyaluronate solution in aqueous saline (0.6% NaCl), 0.1% sodium hyaluronate in aqueous saline (0.9% NaCl) (bonferroni Compared to vehicle treated rats consisting of a two-factor variability assay, p <0.0001, using a Bonferroni multiple comparison test, the tear volume is progressively improved by 6-20 days. In contrast, a drop of 0.1% dexamethasone twice daily showed no sufficient effect on tear volume.

FIG. 8 shows the stability of the tear film with treatment with scopolamine and exposure to dry air, with a significant decrease in the first 3 days, followed by a gradual decrease up to 21 days, as measured by the tear film destruction test. Induces a decrease. Administration of the tresperimus twice daily with 1% drop was performed from 7 days to 21 days compared to vehicle treated mice prepared with 0.1% solution of sodium hyaluronate in saline (0.9% NaCl) (p <0.0001). To significantly improve the stability of the tear film; On the other hand, dexamethasone (dexamethasone) shows a moderate effect that does not continue only on 21 days.

In conclusion, these results show that topical application of tresperimus has an effect on eye dryness by increasing tear secretion and stability of the tear film, two characteristic clinical parameters of eye dryness. These results demonstrate the effect of tresperimus drops on the treatment of clinical signs of dry eye.

Example  3: diabetic retinopathy

Laser photocoagulation is still the standard treatment and vitrectomy is used in the case of retinal detachment. However, a significant proportion of subjects are resistant to laser photocoagulation, and over time, retinal pigment epithelial atrophy associated with the laser wound often progresses under fovea causing reduced vision. Ranibizumab has recently been approved for the treatment of macular edema, but other anti-VEGF agents (bevamizubab) are used without authorization. Combination treatment with anti-VEGF agents can delay laser treatment.

It is possible to note the regression of corticosteroid macular edema and neovascularization. However, side effects are frequent (hypertension, cataracts, endophtalmitis); Moreover, long-term efficacy in diabetic macular edema has not been demonstrated compared to laser treatment.

The effect of Tresperimus is generally assessed in rats using the streptozotocin-induced type I diabetes model, a model of diabetic retinopathy generally described. This Lett model mimics human disease by inducing hyperglycemia associated with the destruction of beta-cells of interest, in which cells produce hormonal insulin, which normally regulates glyceemia. Although there are vascular changes in this model, vasculopathy does not progress to angiogenesis as observed in humans.

Streptozotoxin is injected intravenously into bundled rats. Hyperglycemia progresses rapidly for 5 days after streptozotoxin treatment. Triple after diabetes induction, the levels of VEGF and inflammatory biomarkers are determined in the vitreous.

Oscillatory potentials, as well as electroretinogram (ERG) measurements of a- and b-waves, are analyzed by monitoring photoreceptor damage. The results are compared between the control group of non-diabetic rats treated with tresperimus or vehicle and the group of diabetic rats.

Materials and methods

I. Induction of Diabetes by Streptozotoxin

Diabetes is induced in Sprague Dawley (SD) let (200 g) after overnight fasting by a single 60 mg / kg intravenous injection of streptozotoxin (Sigma) in sodium citrate buffer, pH 4.5. Control non-diabetic animals are treated only with citric acid buffer. After 5 days, animals with 5 g / L of glycemia are considered diabetes.

1. Inflammatory Biomarkers

Three weeks after the induction of diabetes by streptozotoxin, the eye of the let is cut and the vitreous is separated. Some inflammatory biomarkers are measured using the Multiplex Luminex Assay Kit (VEGF, MCP-1, ICAM-1, IL-6, IL-1 Beta; Procarta) according to the manufacturer's recommendations.

2. Electroretinography (ERG)

Diabetic rats are acclimated to cows overnight before ERG testing using LKC's Retinal Potential Recording Method. A series of cow-adapted intensity responses are recorded using a series of Ganzfeld flashes to obtain a rod-mediated retinal response. The individual ERG waveform components (a- and b-waves, flickers) and the amplitude and latency of the vibration potentials are measured by conventional methods.

II. result

The effect of Tresperimus is evaluated in an experimental model of diabetic retinopathy induced by Streptozocin in SD rats. Streptozosin destroys beta cells of interest and thus induces hyperglycemia, a simulated type 1 diabetes. Retinas of diabetic animals show biochemical and electrophysiological abnormalities associated with inflammation. Drops of tresperimus administered twice daily for two weeks at a dose of 0.2% (m / m) in a 0.1% solution of sodium hyaluronate in saline (0.9% NaCl) were dissolved in saline (0.9% NaCl). It does not change blood glucose or body weight as compared to diabetic rats treated with a vehicle made with a 0.1% solution of sodium laurate.

Cytokine and chemokine levels are assessed on samples of vitreous using the multiplex Luminex assay technology. The results reported in FIG. 9 show that MCP-1 and IL-6 levels in the vitreous medium are dramatically increased three weeks after induction of diabetes mellitus by streptozosin. Two-week treatment with Tresperimus drops at a dose of 0.2% twice daily in both eyes from day 7 to day 21 was a one-factor using the inflammatory process (Dunnett multiple comparison test, p = 0.001). Significantly reduce MCP-1 and IL-6 levels in the vitreous of diabetic rats, suggesting an inhibitory effect on monocyte repair.

At 3 weeks after streptozosin treatment, the ERG test showed that diabetic rats after adaptation to cows decreased in size of the a- and b-waves, abnormalities in oscillatory potentials, and light flashes. Regardless of the intensity of the flicker (Figure 10) shows a great deterioration. The visual cells are two types of photoreceptors affected by hyperglycemia. FIG. 10 shows two weeks post-treatment with administration of an eye drop of 0.2% tresperimus twice daily, tresperimus compared to control batch (diabetes treated with vehicle), and the size of flickers It also significantly improves the magnitude of the a- and b-wave and vibrational potential, which implies the neuroprotective effect of the cell in the retinal function, mainly diabetic rats.

Thus, in conclusion, these results show that topical administration of tresperimus is effective against the retina of diabetic rats by reducing retinal inflammation levels and protecting the neuro-retinal function, mainly visual cells, from hyperglycemia. Thus, these results demonstrate the effectiveness of tresperimus drops for the treatment of diabetic retinopathy in humans and for the protection of visual impairment in diabetic subjects.

Claims (25)

Compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof for use in the treatment and / or prevention of inflammatory eye diseases:
[Formula 1]

here,
n is 6 or 8,
A is a bond, group CH ₂ , group CH (OH), group CHF, group CH (OCH ₃ ), group CH ₂ NH or group CH ₂ O,
R is H or CH ₃ .
The method according to claim 1,
The compound may be N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester or Compound which is characterized in that the pharmaceutically acceptable salt.
The method according to claim 2,
The compound is N- [4-[(3-aminopropyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester, tri -A hydrochloride compound.
The method according to claim 1,
The compound may be N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester or Compound which is characterized in that the pharmaceutically acceptable salt.
The method of claim 4,
The compound is N- [4-[(3-aminobutyl) amino] butyl] -carbamic acid, 2-[[6-[(aminoiminomethyl) amino] -hexyl] amino] -2-oxoethyl ester, tetra -A hydrochloride compound.
A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of non-infected uveitis.
A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of severe conjunctivitis, such as spring keratoconjunctivitis.
A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of dry eye syndrome.
A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of diabetic retinopathy.
A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, administered as eye drops.
A compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, administered in an injectable or implantable system.
` Claims administered as a combination of anti-VGEF and anti-TNF agents, corticosteroids, non-steroidal anti-inflammatory agents, antibiotic or immunosuppressant A compound according to any one of 1 to 11 or a pharmaceutically acceptable salt thereof.
As the sole active substance, a compound as defined in any one of claims 1 to 5, and at least one pharmaceutically acceptable excipient suitable for topical administration, wherein the concentration of the active substance is from 0.001% to 1.5%, preferably Aqueous topical formulations, preferably 0.01% to 1.5%.
The method according to claim 13,
The aqueous topical formulation is essentially in the form of eye drops having a neutral pH.
The method according to claim 14,
The aqueous topical formulation comprises hyaluronic acid or a derivative thereof and / or sodium chloride or glycerol.
As the sole active substance, the compound as defined in any one of claims 1 to 5, and at least one pharmaceutically acceptable excipient suitable for injectable ocular administration, wherein the concentration of the active substance is 0.1 μM to 100 mM, preferably Aqueous injectable formulations, preferably from 1 μM to 10 mM.
18. The method of claim 16,
Wherein said aqueous injectable formulation is an intraocular or perocular formulation.
18. The method of claim 17,
Wherein said aqueous injectable formulation is essentially in the form of an injectable solution in a vitreous with a neutral pH.
The method according to any one of claims 16 to 18,
Wherein said aqueous injectable formulation comprises sodium chloride or glycerol.
18. The method of claim 17,
Wherein said aqueous injectable formulation is implantable.
A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, in particular tress, for the manufacture of a medicament useful for the treatment or prevention of inflammatory eye diseases, in particular uveitis, severe conjunctivitis, dry eye or diabetic retinopathy. Use of Perimus or Anisperimus.
A method of treating or preventing inflammatory eye disease, comprising administering to a subject a therapeutically effective amount of a compound represented by formula 1 according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.
23. The method of claim 22,
The inflammatory eye disease is uveitis, severe conjunctivitis, dry eye or diabetic retinopathy, characterized in that the treatment or prevention of inflammatory eye disease.
The method according to claim 22 or 23,
The compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is a method for the treatment or prevention of inflammatory eye disease, characterized in that administered by eye drops.
The method according to claim 22 or 23,
The compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is a method for the treatment or prevention of inflammatory eye disease, characterized in that administered by injectable or implantable system.

Images